Pyridoxine effect in type I primary hyperoxaluria is associated with the most common mutant allele

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1 Kidney International, Vol. 67 (25), pp GENETIC DISORDERS DEVELOPMENT Pyridoxine effect in type I primary hyperoxaluria is associated with the most common mutant allele CARLA G. MONICO,SANDRO ROSSETTI,JULIE B. OLSON, and DAWN S. MILLINER Mayo Clinic College of Medicine, Mayo Clinic Foundation, Rochester, Minnesota; and Mayo Clinic Hyperoxaluria Center, Mayo Clinic Foundation, Rochester, Minnesota Pyridoxine effect in type I primary hyperoxaluria is associated with the most common mutant allele. Background. Pyridoxine (VB6) response in type I primary hyperoxaluria (PHI) is variable, with nearly equal numbers of patients showing partial to complete reductions in oxaluria, and resistance. Because high urine oxalate concentrations cause stones and renal injury, reduction in urine oxalate excretion is deemed favorable. Mechanisms of VB6 action on hepatic alanine:glyoxylate aminotransferase (AGT), the deficient enzyme in PHI, and VB6 dose response have not been wellcharacterized. Methods. Sequencing or restriction site generating polymerase chain reaction (PCR) was used for c.58 genotyping in 23 PHI patients. Pre- and post-vb6 24-hour urine oxalate excretion and VB6 dose were ascertained by retrospective chart review. Results. There were six c.58 G>A homozygotes (AA), eight heterozygotes (GA), and nine patients lacking this change (GG). Pre-VB6 urine oxalate excretion was 152 ± 39, 23 ± 68 and 26 ± 74 mg/1.73 m 2 /24 hours, respectively, and did not differ [AA vs. GA (P =.7); AA vs. GG (P =.7); GA vs. GG, (P =.47)]. Post-VB6 urine oxalate excretion was normal in AA (pre- vs. post-vb6) (P <.1), partially reduced in GA (P <.1), and unchanged in GG (P =.6). Urine oxalate excretion attenuation was similar for VB6 doses (mg/kg/day) of 1 to 4.9, 5 to 9.9, and 1 to 14.9 in AA (P =.41, P =.28, and P =.11, respectively) and GA (P =.42, P =.39, and P =.3, respectively) during follow-up. Conclusion. Presence of the c.58 G>A allele confers VB6 response in PHI and VB6 doses of 5 mg/kg/day appear sufficient. c.58 genotyping can be used to predict VB6 response and guide treatment in PHI. [c represents cdna sequence where nucleotide position +1 corresponds to the adenine (A) of the translation start codon ATG. Equivalent positions based on 5 UTR nucleotide numbering are as follows: c.58 G>A = G63A (Gly17Arg), c.32 C>T = C154T (Pro11Leu), and c.454 T>A = T576A (Phe152Ile)], yields highest residual AGT activity. To test whether VB6 response might be attributable to this allele, we performed c.58 genotyping. Key words: pyridoxine, primary hyperoxaluria, alanine:glyoxylate aminotransferase. Received for publication August 23, 24 and in revised form November 1, 24 Accepted for publication November 3, 24 C 25 by the International Society of Nephrology Primary hyperoxaluria type I (PHI) (McKusick 2599) is an inborn error of glyoxylate metabolism inherited as an autosomal-recessive trait. Deficiency of peroxisomal liver-specific alanine:glyoxylate aminotransferase (AGT) (EC ) causes a marked increase in hepatic oxalate production [1]. Excess oxalate produced cannot be degraded and is eliminated by renal excretion, ensuing in hyperoxaluria. High urine oxalate concentrations result in calcium oxalate deposition within renal parenchyma or stone formation within the urinary tract, progressive renal injury leading to renal failure, and in late stages, systemic calcium oxalate deposition causing severe morbidity and death [2]. PHI clinical expression is diverse, with variable symptom presentation and age at onset of renal failure. The majority of patients present with symptoms related to urolithiasis during childhood or adolescence. The phenotypic spectrum ranges from nephrocalcinosis and/or renal failure in infancy to urolithiasis in adulthood and renal failure in later life [3, 4]. To date, attempts to correlate this clinical heterogeneity with specific mutations of the gene coding for AGT (AGXT)orresidual AGT catalytic activity have been inconclusive [5 8]. One aspect of phenotypic expression that is of particular therapeutic relevance is pyridoxine (VB6) responsiveness. Since PHI pathophysiology appears due to very high urine oxalate concentrations, with oxalate being a recognized cellular and tissue toxin [9], any amelioration of hepatic oxalate production and hence reduction in urinary oxalate concentrations is beneficial. VB6 administration in daily doses of 2 to 1 mg to reduce oxalate excretion in PHI has been noted in case reports since 1961 [1]. In 1998, based on a literature review, Toussaint [11] concluded that approximately 2% of PHI patients would be expected to completely normalize their urine oxalate excretion in response to VB6, 3% would experience a partial response and 5% would be refractory [11]. Further understanding of VB6 response in PHI has been complicated by small numbers of patients, and a wide range of treatment doses and duration in prior published reports. 174

2 Monico et al: Pyridoxine effect in type I primary hyperoxaluria 175 Though attributed to its role as cofactor of AGT, the molecular basis and precise mechanism for VB6 effect in PHI remain unknown. There are no known clinical, biochemical, or molecular markers allowing identification of patients likely to respond to VB6. Testing of the clinical VB6 response by measuring urine oxalate excretion before and after treatment is helpful in patients with preserved renal function but unreliable in patients with moderate to advanced renal insufficiency. Furthermore, many uncertainties remain regarding VB6 dosing and time required for maximal response and permanence of the observed response and VB6 safety, particularly at higher doses due to potential peripheral sensory neuropathy [12]. A molecular-based method for selection of VB6 responsive PHI patients would facilitate optimal treatment and also provide helpful insights into a number of these unresolved issues. In addition to clinical diversity, phenotypic variability at the enzyme level was recognized early. Danpure et al [13] showed the presence of a unique trafficking abnormality among a subset of patients in whom the AGT protein was mistargeted from its normal peroxisomal location to mitochondria where AGT is metabolically ineffective in disposition of glyoxylate, a key metabolic substrate and direct oxalate precursor. AGT mitochondrial targeting is ascribed to synergistic effects of a cryptic mitochondrial targeting sequence encoded by a common polymorphism, c.32 C>T [14], and inhibition of AGT protein dimerization by cosegregation of this and another change, c.58 G>A [15], the most common mutation in PHI. Of nearly 5 AGXT mutations described [16], the c.32 C>T and c.58 G>A haplotype has been recognized to yield highest expression of residual AGT protein and catalytic activity [17] and hence was selected as the most likely candidate allele potentially responsible for VB6 responsiveness. We here demonstrate an association between the c.58 G>A genotype and VB6 responsiveness in 23 PHI patients. METHODS Participation in this Institutional Review Board approved study was offered to all PHI patients followed at our institution. Patients provided written informed consent. Of 37 participants, 23 from 22 unrelated families had availability of pre- and post-vb6 24-hour urine oxalate excretion. Since in PHI plasma oxalate concentration and tissue oxalate accretion begin to rise steeply when glomerular filtration rate (GFR) falls below 4 ml/min/1.73 m 2 [18], suggesting a reduction in urine oxalate excretion, patients with renal clearance below this range were excluded from the study. Clinical VB6 sensitivity could not be evaluated in 13 patients due to renal failure at presentation (N = 9) or unavailability of urine oxalate excretion before VB6 initiation (N = 4). In one infant only random urine oxalate to creatinine ratios were available. The remaining 23 patients are the subject of this report. PHI was diagnosed by hepatic enzyme analysis confirming AGT deficiency in the patient (N = 17) or affected sibling (N = 2) by marked hyperoxaluria with elevated urine glycolate and normal urine glycerate (N = 1) or by marked hyperoxaluria without secondary causes and homozygosity for a known AGXT mutation (N = 3). Because most patients had uninterrupted VB6 treatment since initially prescribed, pre- and post-vb6 urine oxalate excretion, VB6 duration and dose were obtained by retrospective review of medical records. Timing of initial 24-hour urine oxalate measurement after VB6 commencement ranged from 1 to 14 months. Follow-up VB6 doses and corresponding urine oxalate excretion rates were obtained retrospectively from chart review. The recommended daily allowance for VB6 in children and adolescents is age-dependent, ranging.1 to.3 mg in infancy,.5 to.6 mg for ages 1 to 8 years, and 1. to 1.3 mg during adolescence. The recommended daily allowance in adults is 2. mg. Urine was collected for 24 hours in a container acidified with hydrochloric acid to prevent calcium oxalate crystallization. The specimen ph was verified to be <2. prior to analysis. Urine oxalate was measured using the oxalate oxidase assay (normal <4.5 mg (.46 mmol)/24 hours) [19] and values corrected for surface area to permit pediatric to adult comparisons [2]. Simultaneous measurement of 24-hour urine creatinine verified completeness of each collection. GFR was assessed by creatinine or iothalamate clearance. Data are expressed as mean ± SD. Paired or unpaired Student t test was used for comparisons between groups, as appropriate. c.58 genotyping was performed using a restriction site (Msp I) generating polymerase chain reaction (PCR) and/or by direct sequencing of exon 4. Though this mutation detection method has been published previously [21], the authors designed and tested multiple primer pairs and optimized conditions de novo. Introduction of an alternate site for detection of this sequence change, using Mse I, was also developed. Primer sequences will be provided upon request. Hepatic enzyme analyses were performed in laboratories of Dr. C.J. Danpure and Dr. G. Rumsby, University College London, London, United Kingdom, and by Dr. Marie-Odile Rolland, Hopitaux de Lyon, France, in one patient. RESULTS Patients were grouped according to c.58 G>A genotyping. There were six homozygotes (AA), eight

3 176 Monico et al: Pyridoxine effect in type I primary hyperoxaluria Table 1. c.58 genotype, gender,% mean normal residual alanine:glyorylate aminotransferase (AGT) catalytic activity and immunoreactivity, initial ages, VB6 dose and pre-/post urine oxalate excretion rates, years of follow-up, and number of urine oxalate determinations in 23 patients with type I primary hyperoxaluria (PHI) Initial data Follow-up data %Mean normal Pre-VB6 urine Post-VB6 urine Urine oxalate residual AGT AGT Dose oxalate oxalate measurements Geno- catalytic immuno- Age VB6 mg/1.73 mg/1.73 Years of during Patient type Gender activity a reactivity years mg/kg/day m 2 /24 hours m 2 /24 hours follow-up follow-up 1 AA M Not done Not done AA M Not done Not done AA M Not done Not done AA M 12 Positive AA M 17 Weakly AA F 28 Positive Mean ± SD 19 ± ± ± ± ± ± ± GA F 12 Weakly GA M 11 Weakly GA M Sib of GA M 6 Weakly GA M 17 Weakly GA F 1 25% GA M 13 Weakly GA M 14 Weakly Mean ± SD 1 ± ± ± ± ± ± ± GG F 7 Positive GG F 5 Weakly GG M 11 Negative GG F Not done Not done GG F 11 Weakly GG M 7 Negative GG M Nil Weakly GG F Bx in sib GG F Nil Not done Mean ± SD 8 ± ± ± ± ± ± ± 1.8 a AGT catalytic activity, normal range 19.1 to 47.9 lmol/hour/mg protein except in patients 12 and 21 (normal range 2.75 to 8.38 lmol/hour/mg protein), patient 16 (normal range 17.9 to 38.5 lmol/hour/mg protein), and patient 23 (536 ± 14 lmol/second/kg protein). Percent mean normal AGT catalytic activity = residual measured AGT activity/midpoint of normal range times 1. Bx in sib indicates availability of hepatic enzyme analysis in an affected sibling that is not listed in the table. For each group (AA, GA, and GG), the corresponding mean ± SD is given under each column. To convert urinary oxalate values from milligrams to millimoles per 1.73 m 2, divide by 88. heterozygotes (GA) and nine patients who did not possess this change (GG). Clinical and laboratory findings are listed in Table 1. Renal clearance at initial visit was 9 ± 21 (AA), 87 ± 15 (GA), and 98 ± 26 (GG) ml/min/ 1.73 m 2. Pre-VB6 urine oxalate was 152 ± 39 (AA), 23 ± 68 (GA), and 26 ± 74 (GG) mg/1.73 m 2 /24 hours (Table 1) and did not differ significantly among the groups [AA vs. GA (P =.7); AA vs. GG (P =.7); and GA vs. GG (P =.47)]. Residual AGT catalytic activity was higher in AA vs. GA (P =.5) and GG (P =.3) and comparable in GA vs. GG (P =.7). VB6 dose was similar among the three groups (P =.34, P =.25, and P =.13, respectively). Pre- and initial post-vb6 urine oxalate excretion according to genotype is shown in Figure 1. All AA patients showed near to complete normalization of urine oxalate post-vb6 (pre- vs. post-vb6 urine oxalate) (P <.1), and seven of eight GA patients showed a partial reduction in oxaluria (P <.1). The eighth GA patient showed initial complete reduction followed by a partial reduction during subsequent measurements. By contrast, urine oxalate excretion was nearly 2% higher after VB6 in GG though not statistically significant (P =.6). VB6 doses varied during follow-up. Corresponding urine oxalate excretion rates were available during 6.5 ± 2.3 (AA), 8.4 ± 5.8 (GA), and 13.6 ± 11.8 (GG) years of follow-up. VB6 dose ranges (mg/kg/day) of 1 to 4.9, 5 to 9.9, and 1 to 14.9 showed similar urine oxalate reduction in AA (P =.41, P =.28, and P =.11, respectively) and GA (P =.42, P =.39, and P =.3, respectively) (Fig. 2). No further decrease in urine oxalate excretion was observed with doses >5 mg/kg/day in AA or GA patients. In patients lacking the c.58 G>A mutation (GG), urine oxalate was lower at VB6 doses of 5 to 9.9 vs. 1 to 4.9 (P <.1) but remained markedly elevated. Urine oxalate did not decrease further with doses >1 (P =.2). Complete (AA) VB6 response was defined as urine oxalate excretion rate that became normal or near normal (.5 to.7 mmol/1.73 m 2 /24 hours) while the patient was receiving VB6. Partial (GA) VB6 response was defined as a reduction in urine oxalate excretion of 3% or more after administration of VB6. Of the patients receiving the highest initial supplemental dose of VB6, and those

4 Monico et al: Pyridoxine effect in type I primary hyperoxaluria 177 mg/1.73m 2 /24 hr P <.1 P <.1 P =.6 AA GA GG cdna nucleotide 58 genotype mmol/1.73m 2 /24 hr Fig. 1. Pyridoxine effect by genotype in type I primary hyperoxaluria (PHI). Urine oxalate excretion rates before ( ) and after ( ) VB6 administration in patients homozygous (AA), heterozygous (GA), and lacking the c.58 G>A allele (GG). Normal range for urine oxalate excretion is <4.5 mg (.46 mmol)/24 hours. Vertical bars indicate standard deviation. mg/1.73m 2 /24 hr AA GA GG cdna nucleotide 58 genotype * mmol/1.73m 2 /24 hr Fig. 2. Pyridoxine dose response by genotype in type I primary hyperoxaluria (PHI). Urine oxalate excretion rates on VB6 doses (mg/kg/day) of 1. to 4.9 ( ), 5 to 9.9 ( ), and 1 to 14.9 ( )inpatients homozygous (AA), heterozygous (GA), and lacking the c.58 G>A allele (GG). Numbers below each bar indicate the number of measurements for each dose and genotype. Vertical bars indicate standard deviation. remaining on the vitamin for longer than a decade (Table 1), only one patient [14] developed symptoms suggestive of VB6 toxicity (paraesthesias). Symptoms resolved following VB6 discontinuation. DISCUSSION That VB6 administration is able to reduce urine oxalate excretion, and presumably hepatic metabolic production in some, but not all PHI patients, has been known for over 4 years. Predictability of the response and the dose required for therapeutic effect, however, have been unknown, and persistence of the response has been questioned. Here, we demonstrate an association between the c.58 G>A genotype and clinical VB6 responsiveness, establishing the first application of pharmacogenomics in this disorder. VB6 action on hepatic oxalate production, and in turn on renal oxalate excretion, has been ascribed to its role as essential cofactor of the hepatic transamination of glyoxylate to glycine catalyzed by AGT [22]. Whether pyridoxal 5 phosphate, its active form, bestows a favorable effect on human mutant AGT protein via conformational, transcriptional, or prosthetic roles is not known. The potential contribution of other VB6-mediated enzyme systems capable of metabolizing glyoxylate, such as glutamate:glyoxylate aminotransferase in cytosol of hepatocytes, or elsewhere as in renal tubular epithelial cells, is also not well-defined. Regardless of the precise mechanism, the ability of VB6 to favorably impact glyoxylate metabolism is evident in the partial reductions to normalization of oxaluria observed in 61% (14/23) of our patients. The clinical VB6 response was found to be proportional to the number of copies of the c.58 G>A allele. Homozygosity and heterozygosity for this change appear to confer complete and partial responses, respectively, an observation that may be explained by the higher residual AGT catalytic activity coded for by the A allele [17]. The AGT mitochondrial mistargeting mechanism established to date is based on the observation that inheritance of the c.58 G>A and c.32 C>T changes on the same allele causes synergistic inhibition of AGT dimerization which favors entry of AGT monomers into mitochondria [15]. This functional effect is referred to as synergistic because neither change alone is able to reroute AGT protein to mitochondria in vitro [23, 24]. Mitochondrial protein import requires unfolded monomers whereas peroxisomal matrix proteins are generally folded in the cytosol and transported as fully folded tertiary structures. Indeed, Leiper et al [24] specifically showed that AGT dimerization in the cytosol precedes peroxisomal import. Interestingly, salvage pathway molecular chaperones have not been directly observed within mammalian peroxisomal matrix, only along the membranes of these organelles [25]. Brocard et al [25] recently showed that heat shock protein 7, a cytosolic molecular chaperone, can be imported into peroxisomes along with unfolded peroxisomal matrix proteins, challenging the more uniformly held belief that peroxisomal matrix proteins are typically imported into peroxisomes fully folded. These findings have potential implications for the mechanism of action of VB6 and for therapeutic targets in PHI. Pharmacologic doses of the cofactor could rescue mutant AGT protein from being targeted to mitochondria in a process akin to conformational effects seen with chemical and pharmacologic chaperones in endoplasmic reticulum [26]. AGT protein monomers could enter peroxisomes unfolded and fully dimerize into a functional 392-residue homodimer along with its cofactor once inside. However, in an in vitro expression system of a mutant AGT construct containing the c.58 G>A and c.32 C>T changes in COS cells, neither pyridoxine

5 178 Monico et al: Pyridoxine effect in type I primary hyperoxaluria nor pyridoxal phosphate corrected the AGT mistargeting defect [23]. Conversely, glycerol and lowering the temperature, both deemed to have a nonspecific chemical effect on the stability of protein structure, satisfactorily corrected AGT targeting from mitochondria to peroxisomes. Moreover, unlike the in vivo situation whereby the AGT protein appears to always be expressed and catalytically active when coded by this mutant allele, similar in vitro experiments in Escherichia coli have yielded predominantly aggregated, catalytically defunct AGT protein [15]. Additional in vitro studies, perhaps using other expression systems, are needed to further characterize the mechanism of VB6 action in PHI. Data regarding longevity of clinical response to VB6 is scarce. In our cohort, AA and GA patients sustained their respective favorable responses over the entire follow-up period. A single GA patient (Patient 12) initially showed a complete response but then maintained partial VB6 responsiveness during 3 years of follow-up. Factors influencing this unusual pattern of VB6 response are not known and require further investigation. Notably, however, though a loss or gain of VB6 response over time has been reported in some patients [abstract; Leumann Eetal,Proceedings from the Oxalosis and Calcium Oxalate Stone Disease, presented by the National Institute of Diabetes and Digestive Kidney Diseases, in cooperation with the Oxalosis and Hyperoxaluria Foundation, p 33, 2], we did not observe any marked variability, even after many years of follow-up. Prospective dose response studies are needed in AA and GA patients to more precisely define the minimum effective dose of VB6. Such information would be beneficial to long-term patient management. Our results indicate that c.58 G>A genotyping can be used to predict VB6 response and guide treatment in PHI. In AA and GA, VB6 imparts lasting reduction in oxaluria and patients with these genotypes should receive VB6 treatment. VB6 doses of 5 mg/kg/day appear sufficient to achieve sustained normalization of oxaluria in AA and a partial reduction in GA, and hence higher doses do not appear necessary. Whether this effect is due to higher residual enzyme activity or mitochondrial AGT mistargeting per se is not certain. Evaluation of VB6 dose response in patients with other mutant alleles capable of yielding detectable mitochondrial AGT expression (c.454 T>A) or significant residual AGT activity will be of interest. We have detected the c.454 T>A change in a single GG patient from our cohort and her observed clinical response to VB6 resembles that of patients with c.58 G>A heterozygosity. Pending characterization of VB6 effect in patients with other AGXT mutations, a treatment trial is recommended in all GG patients. Due to uncertainties regarding VB6 efficacy and safety in the higher dose range [13], further insights are needed prior to implementing daily doses exceeding 1 mg/kg/day. The demonstration of an association between the c.58 G>A genotype and VB6 responsiveness opens the door to informed use of this valuable agent in the treatment of PHI. ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health (1 KO8 DK ) and the Oxalosis and Hyperoxaluria Foundation. We gratefully acknowledge Ms. Monica I. Poncelet and Mrs. Joanne M. Zimmerman for secretarial assistance. Reprint requests to Carla G. Monico, M.D., Mayo Clinic College of Medicine, 2 First Street SW, Rochester, MN monico.carla@mayo.edu REFERENCES 1. DANPURE CJ, JENNINGS PR: Peroxisomal alanine:glyoxylate aminotransferase deficiency in primary hyperoxaluria type I. 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